Recently, the harmful effects of obesity on health have become a problem. Long-term or invasive treatment is required to promote and sustain weight loss. According to the U.S. Department of Health and Human Services, approximately 300,000 deaths a year in the United States are associated with obesity, and the direct and indirect costs attributed to obesity exceed $100 billion.
Heretofore, anorectic agents such as sibutramine, phentermine, and orlistat have been commercially available; however, they have unsatisfactory appetite-suppressing effects.
There has been growing expectation in recent years that natural peptide hormone, effective for regulating appetite, will be used in medical applications. Several pharmaceutical companies have started clinical trials and preclinical trials regarding the role of natural peptide hormone in obesity.
In particular, a peptide compound PYY3-36 (hereinafter, sometimes abbreviated as “PYY3-36”) represented by the following amino acid sequence: H-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-X (SEQ ID NO: 1), wherein X is OH or a carboxy acid-protecting group, has been receiving attention as a natural peptide hormone. Intranasal administration of PYY3-36 has already been offered as a novel obesity treatment method. PYY3-36 is included in secretions (hereinafter, sometimes referred to as a “secretion”) produced by endocrine cells of the human gut, particularly the stomach. The secretion is readily released based on human food intake, etc.
The secretion usually includes a peptide compound PYY1-36 (hereinafter, sometimes abbreviated as “PYY1-36”) represented by the following amino acid sequence: H-Tyr-Pro-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH2 (SEQ ID NO: 2). PYY1-36 is a compound in which “Tyr-Pro-” is further bound to the N-terminal of PYY3-36, and has a similar amino acid sequence to PYY3-36.
When trypsin is present in the secretion, the trypsin may cleave PYY1-36 and PYY3-36, which generates new peptide digests.
Pharmaceutical formulations containing PYY3-36 can be made, for example, by extracting PYY3-36 from the secretion. However, PYY1-36, and peptide digests derived from PYY1-36 or PYY3-36 may also be present in the pharmaceutical formulations during production.
When the pharmaceutical formulation containing PYY3-36 is in solid form (tablets, powders, etc.), PYY3-36 may be degraded due to long-term storage, which results in the presence of peptide digests derived from PYY3-36 in the pharmaceutical formulation. It has been known that a solid peptide compound can be less stable than a compound in a solution (Non-patent Documents 1 to 4). In a solid peptide compound, a peptide bond is easily cleaved (Non-patent Document 5). Especially, when a peptide compound includes an arginine residue therein, the bond between the arginine residue and the amino acid residue adjacent thereto is easily breakable (Non-patent Document 6).
Therefore, in the PYY3-36-containing pharmaceutical formulation, it is important to determine the presence or absence of PYY3-36, i.e., the presence or absence of peptide digests derived from PYY3-36 during production or after long-term storage. Additionally, when a pharmaceutical formulation is made by extracting PYY3-36 from a secretion, it is preferable to determine the presence or absence of PYY1-36 and peptide digests derived from PYY1-36 during production.
Examples of the determination method include an HPLC method and electrophoresis.
However, PYY3-36, PYY1-36, and the peptide digests thereof have a common amino acid sequence from the C-terminal; therefore, it is difficult to determine the presence or absence of PYY3-36 using known methods. Further, the determination of the complete amino acid sequences of all peptide compounds contained in a pharmaceutical formulation is both time consuming and complicated.
Non-Patent Document 1:
Pearlman, R.; Nguyan, T. J., J. Pharm. Pharmacol. 1992, 44, 178
Non-Patent Document 2:
Strickley, R. G.; Visor, G. C.; Lin, L.; Gu, L., Pharm. Res. 1989, 6, 971
Non-Patent Document 3:
Bhatt, N.; Patel, K.; Borchart, R. T., Pharm. Res. 1990, 7, 593
Non-Patent Document 4:
Jordan, G. M.; Yoshioka, S.; Terao, T., J. Pharm. Pharmacol. 1994, 46, 182
Non-Patent Document 5:
Lai, M. C.; Topp, E. M., J. Pharm. Sci. 1999, 88, 489
Non-Patent Document 6:
Kertscher, U.; Bienert, M.; Krause, E.; Sepetov, N. F.; Mehlis, B., Int. J. Pept. Protein Res. 1993, 41, 207